The present invention relates to the biodegradation of dinitrotoluene in water and soils using microorganisms.
Dinitrotoluenes (DNT) are intermediates in the production of the explosive 2,4,6-trinitrotoluene (TNT) and precursors of toluenediisocyanate, which is used in the manufacture of polyurethane foams. Typically, synthesis yields 76% 2,4-DNT, 19% 2.6-DNT and small amounts of other isomers. Disposal practices associated with TNT manufacturing dating back to World Wars I and II, have resulted in an enormous contamination problem at ammunition production and handling facilities worldwide. The estimated cost for cleanup of defense sites in the United States alone is $2.66 billion. Explosives contamination just at the active installations is estimated to include 750,000 cubic yards of soil and 530 billion gallons of groundwater. Although TNT is no longer produced in the U.S., the current worldwide production of DNT is 2.7 billion pounds per year, some of which ends up in wastewater discharged from DNT manufacturing plants. Both 2,4- and 2,6-DNT are listed as U.S. Environmental Protection Agency (EPA) priority pollutants in, for example, the Clean Water Act and the Safe Drinking Water Act.
2,4-DNT and 2,6-DNT exhibit acute toxicity and low level carcinogenicity. The 14-day LC.sub.50 for guppy (Poecilia reticulata) of 2,4-DNT (69 .mu.M, 12.5 mg/L) and of 2,6-DNT (98 .mu.M, 18 mg/L) indicate aquatic toxicity at low concentrations. EPA treatment standards are 0.32 mg/L for 2,4-DNT and 0.55 mg/L for 2,6-DNT.
Because of the hazards DNT, TNT, and their transformation products pose for drinking water supplies, efforts have been undertaken to understand the fate of the compounds in the environment. DNT may undergo a variety of transformations undeir environmental conditions. One of the most commonly reported transformations has been nonspecific reduction of one or more of the nitro groups attached to the benzene ring to form aminonitro- and diamino-toluenes via hydroxylamino- and nitroso-intermediates. It has also been reported that acetylation is a significant reaction of arylamines produced by reduction of DNT. The reduced intermediates are believed to be responsible for much of the toxicity attributed to DNT. The nonspecific reductions are generally the result of cometabolism, a process in which the transformation of the chemical is incidental to the degradation of another chemical. The organism does not normally derive any metabolic benefit from cometabolic transformations and cannot grow on the cometabolized compound. Cometabolic systems are difficult to control and do not completely destroy the contaminants. Continued transformation of the compound requires input of another carbon source to provide energy to drive the reaction and to keep the necessary enzymes induced.
Mineralization on the other hand allows the organism to use the contaminant as a source of carbon and energy for growth. In mineralization, degradation of the compound goes beyond one or two transformation steps to the cleavage of the carbon backbone, so that the organism derives energy and structural material from the compound, and releases small inorganic molecules, predominantly water and CO.sub.2.
Because the bacteria derive a benefit, mineralization of the contaminants results in a controllable self sustaining process. Mineralization of 2,4- and 2,6-DNT at concentrations up to 10 mg/L by natural river water populations collected downstream of a TNT-manufacturing plant, following lag periods of up to 3 weeks, has been reported. Neither the pathways involved nor the organisms responsible for the mineralization were characterized. Based upon rate studies, it was concluded that at low DNT concentrations, microbial degradation played only a minor role in the removal of DNT from surface waters. Mineralization of 2,4- and 2,6-DNT by populations of microorganisms indigenous to a shallow aquifer at an explosives-contaminated site have also been reported. In all cases, DNT concentrations were less than 20 mg/L. After 28 days, 28% of initial 2,4-DNT was mineralized, 20% was undegraded, 28% was recovered as aminonitrotoluenes and 24% was in unidentified metabolites. For 2,6-DNT the percentages were 8, 67, 14, and 11%, respectively. The picture that emerged from these reports of mineralization of DNT by natural assemblages was one of slow mineralization, which, even under aerobic conditions, lost out to competition from nonspecific transformation reactions.
Extensive research has focused on the microbial transformation of nitroaromatic compounds and how contaminated sites can be cleaned up through bioremediation. Removal of DNT by microbial cultures has been reported; however as stated above, processes used to date have generally been cometabolic, and the microorganisms involved did not use DNT as a growth source. Mixed cultures that cometabolize 2,4-DNT under anaerobic conditions when ethanol is provided as the primary carbon and energy source have been reported. In these systems aromatic amines accumulated. The major strength of the anaerobic system is the ability to transform 2,4-DNT in the presence of other carbon sources.
Composting systems for explosives-contaminated soils have received much attention in recent years, however no composting systems have been developed specifically for DNT. At present, composting presents two major drawbacks: (1) generally, no more than 10 to 30% of the compost pile can be made up of the contaminated soil, resulting in an increase in the amount of product that must eventually be landfilled; and (2) despite concerted efforts, residual toxicity of finished compost is still undetermined. It has been reported that fungi mineralize DNT under ligninolytic conditions. However, the ligninolytic systems do not appear to be involved in the initial reduction of DNT, which requires live mycelia. The ligninolytic system is necessary for the mineralization of the aminoaromatic products of DNT reduction. The limiting factor in the fungal systems has been the toxicity of DNT at concentrations found in contaminated soils.
Bacterial strains capable of degrading 2,4-DNT have been isolated and characterized and the degradation pathway determined. The pathway proceeds through dioxygenation of 2,4-DNT to 4-methyl-5-nitrocatechol; monooxygenation of 4-methyl-5-nitrocatechol then yields 2-hydroxy-5-methylquinone which is subsequently reduced to 2,4,5-trihydroxytoluene prior to ring cleavage. 2,4-DNT-degrading bacteria are not, however, able to degrade the mixtures of DNT isomers (80:20 2,4-DNT:2,6-DNT) found at contaminated sites, and the presence of high concentrations of 2,6-DNT can inhibit 2,4-DNT degradation. Cultures grown on 2,4-DNT, when incubated in the presence of mixtures of 2,4-DNT and 2,6-DNT, are inhibited in the degradation of 2,4-DNT, and 4-methyl-5-nitrocatechol accumulates in the culture fluid, as well as a metabolite from 2,6-DNT.
It is an object of the present invention to provide a process for the biodegradation of samples contaminated with a mixture of 2,4-dinitrotoluene and 2,6-dinitrotoluene.
Other objects, aspects and advantages of the present invention will be apparent to those skilled in the art from a reading of the following detailed disclosure of the invention.